Method for the preparation of biopolymers

ABSTRACT

The present invention relates to a method of preparing and separating biopolymers and biopolymer fractions useful for wastewater treatment applications from sewage sludge comprising the steps of disrupting the bacterial cell walls of bacteria present in the sewage sludge by at least 75% to release the intracellular contents of the bacterial cells and separating the biopolymers from any contaminants present.

The present invention relates to a method of preparing and separatingbiopolymers and biopolymer fractions from sewage sludge. In particular,the present invention relates to the preparation and separation ofbiopolymers and biopolymer fractions which are derived frommicro-organisms, especially micro-organisms or biomass arising fromwastewater treatments. Such wastewater treatments include for exampleactivated sludge processes which are commonly used for the treatment ofmunicipal sewage.

Such sludges may contain biopolymers or biopolymer fractions in the formof peptidoglycan or deoxyribonucleic acid (DNA) fractions or acombination of these two materials.

The biomass which arises from the treatment of wastewater generated inhouseholds, commercial properties and industry is commonly known assewage sludge. That is, the term sewage sludge refers to sludge whichhas not been subjected to a digestion stage, whilst biological sludge isa sludge that is generated from the biological treatment of sewage.

In Europe, the treatment of sewage sludge follows the Urban WastewaterTreatment Directive (91/271/EEC). Annual sludge production currentlystands at 1.3 M tonnes for the UK and 10M tonnes for Europe. Globally,the volume of sludge is expected to increase significantly over thecoming years as many of the developing economies start to implementtheir own pollution control measures. Consequently, sewage sludge willneed to be either:

-   -   i) treated to provide a source of energy and valuable raw        materials which may be recovered for useful applications; or    -   ii) destroyed to prevent pollution.

Presently there are only two practical options for sludge management,namely agricultural recycling or incineration. Agricultural recycling isregarded as the best practicable environmental option. Incineration, onthe other hand, is much more costly and socially objectionable. However,incineration is still preferred due to the possible risks arising withagricultural recycling.

The most common method of sludge treatment is digestion, specificallyanaerobic digestion, which recovers the energy content of the sludge asbiogas and reduces the odour and pathogen level of the sludge to make itsuitable for agricultural recycling. In general, anaerobic digestioncomprises a series of complex biochemical reactions mediated by aconsortium of micro-organisms that convert organic compounds intomethane and carbon dioxide. Anaerobic digestion is also a stabilizationprocess, achieving odour, pathogen, and mass reduction.

The digested sludge residue arising from anaerobic digestion primarilycomprises various types of bacteria and hence bacterial cells. Bacterialcells represent a huge untapped resource globally.

Most bacteria in sludge are gram-negative, and in activated sludgeaccount for over 90% of the bacteria strains. Gram negative bacteriapossess a relatively ‘thin’ cell wall consisting of only a few layers ofpeptidoglycan which comprises about 10% of the biomass of the bacteria.

Peptidoglycan (also known as murein) is a unique biopolymer whichconsists of both D- and L-amino acids. The basic structure ofpeptidoglycan consists of a carbohydrate backbone of alternatingresidues of β-(1,4) linked N-acetyl glucosamine and N-acetyl muramicacid. Attached to the N-acetyl muramic acid is a peptide chain of 3 to 5amino acids. The peptide chain can be cross-linked to another peptidechain on another carbohydrate strand forming a 3-D mesh-like layer.

In contrast, the intracellular components DNA and RNA account for about23% of the dry mass of a bacteria cell. The remainder of the biomass,that is, the 67% of the dry mass that constitutes the bacteria cell,includes for example polysaccharides, proteins and phospholipids. Thepolysaccharides, proteins and phospholipids which are found largelyoutside the cell wall are often referred to as the extracellularpolymeric substances (EPS). The EPS typically account for 50% of cellbiomass.

Both DNA and RNA are biopolymers composed of repeating units ofnucleotides. Each nucleotide consists of a sugar, a phosphate and anucleic acid base. The bases are hydrophobic and relatively insoluble inwater at the near neutral pH of the cell. At acidic or alkaline pH theybecome charged, and their solubility in water increases. Theinteractions between DNA and metals, particularly heavy metals, havebeen extensively studied. The binding of metals to the nucleic acidsgenerally occurs through the formation of complexes. DNA therefore actsas a biological ligand for metals and may associate with metals aftercell lysis.

Exciting possibilities have been suggested for the use of biopolymersand biopolymer fractions isolated from sewage sludge. For example, ithas been mooted that biopolymers and biopolymer fractions from sewagesludge could be employed as substitutes for polymers and copolymerscommonly used as flocculants/coagulants in municipal and industrialwastewater treatment.

Alternatively, biopolymers and biopolymer fractions have been suggestedas potentially useful products for wastewater treatment and the removalof undesirable contaminants such as heavy metals or valuable commoditiessuch as phosphorus.

However, hitherto there are no known methods for commercial manufactureof biopolymers and the efficacy of their use in any of the suggestedapplications so far has never been demonstrated in practice.

Nevertheless, if new methods were available to enable the biopolymers tobe recovered, preferably in a relatively pure form, the biopolymers andbiopolymers fractions would potentially provide a viable income forsludge producers and, more importantly, a new option for sludgemanagement.

Therefore there exists the need for a suitable method for thepreparation of biopolymers and biopolymer fractions from sewage sludge.

More particularly there exists the need for a method of preparingbiopolymers and biopolymer fractions which also allows the isolation andseparation of biopolymers and biopolymer fractions from sewage sludge.

It is therefore an aim of the present invention to provide a method forthe preparation and separation of biopolymers and biopolymer fractionsfrom sewage sludge which addresses the requirements of the industry.

It is a further aim of the present invention to provide a new andimproved method for the preparation and separation of biopolymers andbiopolymer fractions from sewage sludge which is both effective andefficient and which may be applied to wastewater treatment applications.

According to a first aspect of the present invention there is provided:a method of preparing and separating biopolymers and biopolymerfractions from sewage sludge, the method comprising the following steps:

i) disrupting the bacterial cell walls of bacteria present in the sewagesludge by at least 75% to release the intracellular contents of thebacterial cells; and

ii) separating the biopolymers from any contaminants present.

In step (i) of the method of the invention at least 75% of the totalamount of cells present are ruptured, that is, cells are ruptured by atleast 75% to release their contents. Preferably, the bacterial cellwalls may be disrupted by at least 85% to release the intracellularcontents of the bacterial cells, more preferably by at least 90%, evenmore preferably by 95% and most preferably by 99%. That is, the higherthe degree of rupture the more biopolymers are available for recovery.

In step (i) the intracellular contents of the bacterial cells arereleased so as thereby to free soluble biopolymers in the form of a celllysate. The biopolymers separated by the process may be solublebiopolymers in the cell lysate or may be insoluble biopolymers presentin the cell lysate.

The method of the present invention may further comprise the step of:removing extracellular polymeric substances (EPS) from the bacterialcells in the sewage sludge prior to disrupting the bacterial cell walls.

The method of the present invention may also further comprise the stepof: precipitating the soluble biopolymers from the cell lysate prior toseparating the biopolymers from any contaminants present. The solublebiopolymers precipitated preferably comprise nucleic acids.

Alternatively, the cell lysate may be anaerobically digested todecompose nucleic acids so that insoluble peptidoglycan present in thecell lysate may be separated and isolated.

The method of the present invention may preferably be used with knownand readily available equipment which requires minimal cost toimplement.

The method of the present invention is therefore also suitable for largescale industrial biopolymer production in quantities commensurate withthe operation of a municipal sewage treatment works and thereforeprovides an answer to the problem of how to manage the ever increasingproblem of sewage waste.

It will be understood that the term ‘sludge’ referred to herein refersto the solid fractions found in wastewater and sewage and which arisefrom the treatment of the wastewater and sewage.

Typically in the treatment of municipal sewage the wastewater issubjected to a sedimentation process wherein a certain amount ofsuspended solids settle out under the effect of gravity yielding a firstsludge fraction known as ‘primary sludge’.

The settled wastewater containing mainly dissolved organic substancesand nutrients is subsequently treated biologically in a second processcommonly known as the ‘activated sludge process’.

Sludge from the second process is known as ‘biological sludge’ which ismostly made up of bacterial cells whereas the primary sludge containsmainly food residue and other inert inorganic components such as siltand fine sand.

The biological sludge fraction and primary sludge fraction are oftencombined to provide combined sludge or co-settled sludge. The combinedsludge from the treatment of municipal sewage typically contains between40 to 60% weight/weight (w/w) of biological sludge. A typical sludgematrix may therefore comprise 35% biological sludge, 35% primary sludgeand 30% inert inorganic matter on a dry weight basis.

It is preferred that in the method of the present invention the sewagesludge is a biological sludge, i.e. the sludge obtained by means of thesecond process known as the activated sludge process.

In addition, in relation to the method of the present invention theruptured bacterial cell walls may be ground to disperse or dissolve thepeptidoglycan.

The extracellular polymeric substances (EPS) may be removed using amechanical means, or alternatively the EPS may be removed using chemicalmeans or enzyme treatments. For example, the EPS may be dislodged fromthe cell wall by treatment with a shearing device or a low powerultrasonic device before anaerobic digestion.

Also in relation to the method of the present invention the cell wallsmay be disrupted using one or more of: ultra-sonication, and/or beadmills and/or caustic treatments. For convenience, cell rupture should beperformed after EPS removal to minimise contamination of thebiopolymers.

It is preferred that in the method of the present invention thepeptidoglycan present in the cell lysate is separated and isolated fromthe cell DNA.

When peptidoglycan is the desired product, the cell lysate is preferablyanaerobically digested to destroy the DNA and in addition, the celllysate is preferably filtered after digestion to recover thepeptidoglycan.

It is known in the art that large molecules such as biopolymers havesurfaces and their surfaces can have affinity for water or oil orcertain chemicals. The nature of the surfaces of the biopolymers isreferred to as their surface properties. In relation to the method ofthe present invention the biopolymers and fractions may be treatedbefore, during or post the precipitation step with one or more chemicalsto alter or enhance the surface property of the biopolymers and improvethe binding of specific compounds to the biopolymers and fractions.

The DNA and/or peptidoglycan isolated using the method the presentinvention may be used as flocculants and coagulants and as a replacementfor known flocculants and coagulants.

According to a second aspect of the present invention there is provideda method for the preparation and separation of biopolymers andbiopolymer fractions from sewage sludge, the method comprising thefollowing steps:

i) removing extracellular polymeric substances (EPS) from bacteria cellsin the sewage sludge;

ii) disrupting the bacterial cell walls by at least 75% to release theintracellular contents of the bacterial cells;

iii) precipitating soluble biopolymers to form a cell lysate andseparating the biopolymers from any contaminants present.

According to a third aspect of the present invention there is providedthe use of the DNA and/or peptidoglycan isolated using the methodaccording to the first or second aspect of the present invention asflocculants and coagulants.

The biopolymer preparation using the methods according to the presentinvention enables the recovery of biopolymer in a form that is pure,active, and in a high yield.

The term ‘active’ referred to in relation to the biopolymer means thatthe biopolymers are not damaged or degraded in any way which may degradethe functions of the biopolymers.

From the foregoing it will be clear that biological sludge is the beststarting material for biopolymer production since primary sludge is nota biopolymer source and would only add to the contaminant load which hasto be removed. Nevertheless, for operational reasons, it may be moreexpedient to start the biopolymer preparation process with combinedsludge as will be demonstrated later. In contrast, the preparationprocess is considerably simplified however where the biological sludgeprovides the feed stock for the biopolymer.

The term biopolymer fraction referred to herein relates to a fraction ofbiopolymers which have a similar molecular size or surface propertytaken from a population of biopolymers.

The purity of a biopolymer fraction may be defined as follows:

Purity=mass of biopolymer/total mass of the product

For biological sludge, the removal of the extracellular polymersubstances (EPS) alone increases the purity of a biopolymer fraction by100%, since the EPS typically account for 50% of the cell biomass. EPSmay be removed by a number of mechanical means such as by using highshear rates. Alternatively, EPS may be removed by chemical means such assurfactant treatments or enzyme treatments. Digestion commonly achieves50% EPS removal.

Numerous techniques are available for cell lysis (or bacterial cell walldisruption/rupture) including: high shear methods, high pressure methodsand high temperature methods; as well as chemical and enzymatic means.

Suitable methods for disrupting or rupturing bacterial cell walls torelease the intracellular content for use with the present inventioninclude: ultra-sonication means, bead mills, and caustic treatment.

It should also be noted that bacterial cell wall disruption only causesthe cells to fracture thereby releasing any intracellular content orexposing the intracellular content to possible chemical attacks, butleaving the crystalline structure of the cell wall relatively intact.

Biopolymer molecules are often partially crystalline (also referred toas semi-crystalline), with crystalline regions dispersed withinamorphous material. Crystalline polymers are denser, more physicallyrobust and more resistant to chemical attacks than amorphous polymers.

It is desirable to dissolve the intracellular contents withoutdissolving or generating very small cell wall fragments in order toconveniently separate the different intracellular materials by forexample filtration.

In contrast, prolonged action on the bacterial cells by a bead mill(also known as a micro-mill) employing small beads of very high hardnesssuch as ceramic beads, further reduces the size of the bacterial cellwall fragments. However, the bacterial cell wall fragments only swell ifexposed to high pH conditions. Therefore, caustic treatment alone is notsufficient to achieve dissolution of the peptidoglycan. The dissolutionof the peptidoglycan requires grinding of the cell fragments in acaustic media with ceramic beads. In contrast, nucleic acids are morereadily soluble in a caustic media and often do not required anygrinding action.

For specific applications, relatively pure DNA or pure peptidoglycanfractions are desirable. That is, DNA fractions at levels of at least50% purity; more preferably at least 70% purity and most preferably atleast 85% purity are desirable.

The separation of DNA from peptidoglycan may be achieved by firsttreating the cell lysate with anaerobic digestion conditions whereby allof the nucleic acids undergo gasification (that is, the nucleic acidsare converted to biogas in the form of methane and CO₂) leaving thepeptidoglycan relatively intact. Alternatively, any cell fragments maybe separated from the soluble nucleic acids by filtering through asuitably rated filter.

It will be appreciated that the cell lysate or filtered lysatecontaining the soluble DNA and/or soluble peptidoglycan may also containother soluble contaminants. Such contaminants may be convenientlyseparated from the biopolymers by selective precipitation of thebiopolymers. For example, both DNA and peptidoglycan are ‘poly-acids’which readily precipitate as the pH of solution drops. DNA andpeptidoglycan are also susceptible to precipitation due to chargeneutralisation with multivalent cations such as Ca++ and Mg++ ions.

DNA and peptidoglycan are also known as macro-molecules and may interactand bind with many different molecules. The ability of themacromolecules to bind different molecules makes the biopolymersvaluable as agents for wastewater treatment.

It is also possible to alter or enhance their surface property toimprove their binding of specific compounds with one or more suitablechemicals. Therefore, with suitable modifications the nucleic acids andpeptidoglycans may be used as for example but not limited to: surfaceactive agents, coagulants, flocculants, adsorbents, surface coatings,complexing agents. Chemical modification of the biopolymers withspecific ligands may be conveniently carried out with staining andintercalating techniques.

In chemistry, intercalation is the reversible inclusion of a molecule(or group) between two other molecules (or groups). Examples include DNAintercalation and graphite intercalation compounds. There are severalways in which molecules (in this case, also known as ligands) mayinteract with DNA. For example, ligands may interact with DNA by:covalently binding; electrostatically binding; or intercalating.Intercalation occurs when ligands of an appropriate size and chemicalnature insert between base pairs of DNA. These ligands are mostlypolycyclic, aromatic, and planar, and therefore often make effectivenucleic acid stains. Examples of DNA intercalators include: berberine,ethidium bromide, proflavine, daunomycin, doxorubicin, and thalidomide.DNA intercalators are used in chemotherapeutic treatment to inhibit DNAreplication in rapidly growing cancer cells.

Staining is an auxiliary technique used in microscopy to enhancecontrast in the microscopic image. Stains and dyes are frequently usedin biology and medicine to highlight structures in biological tissuesfor viewing, often with the aid of different microscopes. Stains may beused to define and examine bulk tissues (highlighting, for example,muscle fibres or connective tissue), cell populations (classifyingdifferent blood cells, for instance), or organelles within individualcells.

In biochemistry there is also the requirement to add a class-specific(DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify orquantify the presence of a specific compound. Staining and fluorescenttagging may serve similar purposes. Biological staining is also used tomark cells in flow cytometry, and to flag proteins or nucleic acids ingel electrophoresis. Certain stains are often combined to reveal moredetails and features than a single stain alone. Combined with specificprotocols for fixation and sample preparation, scientists and physiciansmay use these standard techniques as consistent, repeatable diagnostictools. A ‘counterstain’ is a stain that makes cells or structures morevisible, when not completely visible with the principal stain. Forexample, crystal violet stains only gram-positive bacteria in gramstaining. A safranin counterstain is applied that stains all cells,allowing identification of gram-negative bacteria.

In one embodiment of the present invention there is provided a method ofpreparing a DNA/peptidoglycan blend from sewage sludge in which theblend contains mainly DNA and peptidoglycan and which comprises thefollowing steps:

-   -   1) removing extracellular polymeric substances (EPS) from        bacterial cells in sewage sludge by anaerobic digestion;    -   2) rupturing/lysing the bacterial cell walls by at least 75% to        release the intracellular contents of the bacterial cells using        sodium hydroxide (NaOH) and milling to solubilise the DNA/RNA        and other contaminants present;    -   3) precipitating the DNA onto the peptidoglycan debris using a        suitable precipitant such as calcium chloride (CaCl₂);    -   4) removing the precipitate containing the DNA/peptidoglycan        blend from the process liquor using a suitable solid liquid        separation technique such as sedimentation or centrifugation.

In an alternative embodiment of the present invention there is provideda method of isolating peptidoglycan from sewage sludge which comprisesthe following steps:

-   -   1) optionally removing extracellular polymeric substances (EPS)        from bacterial cells in sewage sludge by anaerobic digestion;    -   2) rupturing/lysing the bacterial cell walls by at least 75% to        release the intracellular contents of the bacterial cells using        sodium hydroxide (NaOH) and milling to solubilise the DNA/RNA        and other contaminants present;    -   3) removing DNA/RNA and other contaminants present including any        EPS present from bacterial cells in sewage sludge by anaerobic        digestion;    -   4) removing the peptidoglycan debris from the digestate using a        suitable solid liquid separation technique such as filtration or        centrifugation.

For a better understanding of the present invention and to show moreclearly how it may be carried into effect, the following examples willnow be discussed below.

EXAMPLE 1

Samples of biopolymer were prepared by milling samples of digestedsludge cake followed by an extraction procedure as follows. (Digestedsludge cake is the sludge solid fraction obtained by substantiallyremoving the water from digested sludge).

A digested sludge cake with a typical dry mass content of 25% was milledusing a Capco Ball Mill 12VS. The samples were processed for three daysin an aqueous caustic environment in order to break down the cellstructure. The milling took place in 500 ml bottles under the followingconditions:

Sludge Cake 50 g (25% Dry Solids) Distilled Water 150 ml NaOH (Dry Mass)greater than 5% Milling Time greater than 48 hours Final pH 9.0 MillingSpeed 100 rpm Milling Media 2 mm ZrO₂ (200 ml)

The biopolymer was precipitated from out of the cell lysate by theaddition of calcium chloride (CaCl₂) solution. A 20% solution of CaCl₂to lysate in a ratio of 1:2 was used. The precipitate resulting fromthis operation was recovered by centrifugation and washed with distilledwater before use as an extracted biopolymer.

The extracted biopolymer had a compacted but granular appearance with adry solid content of 16.97% weight/weight (w/w). Initial analysis showedthat the granules had a phosphorus (P) content of 6.5% weight/weight(w/w, dry basis) after treatment with the 20% solution of CaCl₂.

The extracted biopolymer purity was estimated to have a DNA content of8% weight/weight (w/w dry basis).

EXAMPLE 2

A number of experiments were conducted to demonstrate the use of abiopolymer as an agent for the removal of orthophosphate (OP) fromsettled sewage. The typical OP levels for settled sewage at a test sitewere in the region of 2 mg/L, so for the purpose of the experiments thesettled sewage was spiked with sodium di-hydrogen phosphate to bring theOP content up to a value of 20 mg/L.

A ‘Jar’ test protocol was then used which comprised a test in which to500 mL of settled sewage were added varying amounts of crude biopolymer(in the form of cell lysate) followed by the addition of 20 mL of CaCl₂solution (at 20%) with rapid mixing (200 rpm) for 1 minute. A 15-minuteperiod was allowed for flocculation/coagulation followed by a settlingperiod of 15 minutes before analysis.

Table 1 illustrates the results of contacting the biopolymer in the formof cell lysate with settled sewage. The ‘Jar-test’ protocol indicated avarying degree of orthophosphate removal depending on the amount of thecrude biopolymer (cell lysate) added to the test mixture. The resultsare shown in Table 1 which illustrates the effectiveness of crudebiopolymer in phosphorus removal from wastewater.

TABLE 1 Total Amount of System Residual Total Ca:P Settled cell lysateOP OP System Ratio Sewage added (mL) (mg) (mg/L) Ca (mg) (w/w) OPRemoval % 5 13.3 7.4 103 7.8 62 10 16.8 2.9 206 12.3 85 15 20.3 1.4 30915.2 93 20 23.8 1.4 412 17.3 93 OP—orthophosphate

EXAMPLE 3

The protocol outlined in example 2 was followed except that in example 3the crude biopolymer in the form of cell lysate was replaced by 10 g ofextracted biopolymer and no CaCl₂ was added. The settled sewage was alsoreplaced with sludge liquor with orthophosphate (OP) content up to about100 mg/L and with varying amounts of volatile fatty acids (VFA) present.

The results of the biopolymer trials using the sludge liquor containingorthophosphate are shown in Table 2.

Table 2 illustrates the effectiveness of the extracted biopolymer inphosphorus removal from sludge liquor.

TABLE 2 Sample Residual OP OP Removal Residual Calcium (Added VFA mg/L)(mg P/L) (%) (mg/L) pH 200 11.8 85.0 1542 6.8 300 25.7 72.7 1602 6.3 40037.9 56.5 1472 6.0 500 49.4 48.5 1548 5.6 600 55.0 43.0 1572 5.5

It will be appreciated that many modifications and enhancements may bemade to the basic method and product outlined herein.

For instance, the methods of separating DNA from peptidoglycan or othercontaminants; and of precipitating the biopolymers may be varied, forexample by pH adjustment or by adding salts similar to CaCl₂ or evenorganic solvents.

Other possible modifications will be readily apparent to theappropriately skilled person.

1. A method of preparing and separating biopolymers and biopolymerfractions from sewage sludge, the method comprising the following steps:i) disrupting the bacterial cell walls of bacteria present in the sewagesludge by at least 75% to release the intracellular contents of thebacterial cells; and ii) separating the biopolymers from anycontaminants present.
 2. A method according to claim 1 furthercomprising the step of: removing extracellular polymeric substances(EPS) from the bacterial cells in the sewage sludge prior to disruptingthe bacterial cell walls.
 3. A method according to claim 1 or 2 furthercomprising the step of: precipitating the soluble biopolymers from thecell lysate prior to separating the biopolymers from any contaminantspresent.
 4. A method according to claim 3 wherein the solublebiopolymers precipitated comprise nucleic acids.
 5. A method accordingto claim 1 or 2 wherein the cell lysate is anaerobically digested andpeptidoglycan present in the cell lysate is separated and isolated.
 6. Amethod according to any of claims 1 to 5 wherein the sewage sludge is abiological sludge.
 7. A method according to any of claims 1 to 6 whereinthe ruptured bacterial cell walls are ground to disperse or dissolvepeptidoglycan present therein.
 8. A method according to any of claims 1to 7 wherein the extracellular polymeric substances (EPS) are removedusing a mechanical means.
 9. A method according to any of claims 1 to 7wherein the extracellular polymeric substances (EPS) are removed using achemical means.
 10. A method according to any of claims 1 to 7 whereinthe extracellular polymeric substances (EPS) are removed using enzymetreatments.
 11. A method according to any of the preceding claimswherein the bacterial cell walls are disrupted by means of one or moreof: ultra-sonication, and/or bead mills, and/or caustic treatments. 12.A method according to any of the preceding claims wherein peptidoglycanpresent in the cell lysate is separated and isolated from the cell DNA.13. A method according to claim 12 wherein the cell lysate isanaerobically digested.
 14. A method according to claim 12 wherein thecell lysate is filtered.
 15. A method according to any of claims 1 to 14wherein the biopolymers are treated before, during or post theprecipitation step with one or more chemicals.
 16. A method for thepreparation and separation of biopolymers and biopolymer fractions fromsewage sludge, the method comprising the following steps: i) removingextracellular polymeric substances (EPS) from bacteria cells in thesewage sludge; ii) disrupting the bacterial cell walls by at least 75%to release the intracellular contents of the bacterial cells; iii)precipitating soluble biopolymers to form a cell lysate and separatingthe biopolymers from any contaminants present.
 17. Use of DNA isolatedusing the method of any of claims 1 to 16 as flocculants and coagulants.18. Use of peptidoglycan isolated using the method of any of claims 1 to16 as flocculants and coagulants.